. .,

DETECTION OF L. MONOCYTOGENES IN ANIMAL AND WATER SAMPLES IN KUBAH NATIONAL PARK, KUCHING, SARAWAK

Aimi Syazana. Anuar 23072

QR 201 L7 Al94 Bacbelor of Science witb Honours 2012 (Resource Biotecbnology)

.. 2012

t' 4aSlll Khidmal Maklumat Ak demik I • , ". UNlVERSm MALAYSIA SAKAWA)(

Detection of L monocytogenes in animal and water samples in Kubah National Park, Kuching, Sarawak

Piliiililiiiili~iili' . 1000235737

Aimi Syazana Anuar (23072)

This project is submitted in partial fulfilment of the requirements for the degree of Bachelor of Science with Honours

(Resource Biotechnology)

Department of Molecular Biology

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARA WAK

2012

, .

ACKNOWLEDGEMENTS

This research project would not have been possible without the support of many people.

Above all, I would like to thank God for His blessing upon the completion of this project. I

would like to express my gratitude to my supervisor, Dr. Lesley ,Maurice Bilung who was

abundantly helpful and offered invaluable assistance, support and guidance. Deepest gratitude

is also dedicated to both of co supervisors, Prof. Dr. Kasing Apun and Dr. Samuel Lihan for

sharing their valuable knowledge and experiences. Special thanks to postgraduate students

especially Miss Velnetti Linang and Miss Christy Chan Sien Wei from Microbiology

Laboratory for sharing the literature, methodology, experiences, tips and helpful assistance

throughout this project. Not forgetting to my beloved lab colleagues for providing me the best

working atmosphere for these two semesters. Finally, I would like to thank my beloved

families and friends; for their patience, understanding and endless love, through all the

hardships and joys of this project.

iii

I • Pusat Khidmat Maklumat Akadtmik UNIVERSITI MALAVSIA SARAWAK

TABLE OF CONTENTS

DEC LARA TION ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS ABSTRACT/ABSTRJU(

CHAPTER I

CHAPTER II

2.1

2.2

2.3

2.4 2.5 2.6

2.7

CHAPTER III

3.1

3.2 3.3

3.4

3.5

INTRODUCTION

LITERATURE REVIEW

Selection of species studied

2.1.1 Family Listeriaceae

2.1.2 Genus Listeria

Listeria monocytogenes

2.2.1 Morphology and culture

Factors affecting the growth ofL. monocytogenes

2.3.1 pH 2.3.2 Temperature

Virulence factors

Epidemiology Listeriosis

Antibiotic resistance

MATERIALS AND METHOD

Sample collection

Presumptive Listeria spp. isolation

Isolation ofListeria species

3.3.1 Enrichment ofbacteria sample

3.3.2 Selective isolation ofListeria spp. on P ALCAM

selective agar 3.3.3 Preparation of working culture and stock culture

Biochemical analysis

3.4.1 Gram staining Molecular analysis

3.5.1 DNA extraction

3.5.2 Specific Polymerase Chain Reaction (PCR) to detect

L. monocytogenes using hlyA gene

Page

11

III

IV

VI

V11

Vlll

IX

1

4

5

6

7 8 8 10 11

12

14 17

18

18

19

19

19

20

IV

, .

3.5.3 Agarose gel electrophoresis (AGE) 21 3.6 Antimicrobial susceptibility test 22

3.6.1 Preparation and turbidity standard for inoculums 23 3.6.2 Inoculation of test plate 23 3.6.3 Multiple Antibiotic Resistances (MAR) analysis 24

CHAPTER IV RESULTS

4.1 Isolation ofListeria spp. 25 4.2 Biochemical analysis

4.2.1 Gram staining 28 4.3 Molecular analysis

4.3.1 Specific PCR to detect hlyA gene 30 4.4 Antimicrobia1 susceptibilities ofL. monocytogenes isolates 32

in animal and water samples in Kubah National Park, Kuching, Sarawak

4.4.1 Multiple Antibiotic Resistances (MAR) analysis 35

CHAPTER V DISCUSSION

5.1 The occurrence ofListeria spp. in Kubah National Park, 36 Kuching, Sarawak

5.2 Specific Polymerase Chain Reaction (PCR) 38 5.3 Antimicrobial susceptibilities profiles ofL. monocytogenes 40

in animal and water samples at Kubah National Park, Kuching, Sarawak

CHAPTER VI CONCLUSIONS AND RECOMMENDATIONS 43

REFERENCES 44

v

· ./

LIST OF TABLES

Tables Page

3.1 List ofanimal samples positive for presumptive Listeria spp. 16

from Kubah National Park, Kuching, Sarawak

3.2 List ofwater samples positive for presumptive Listeria spp. 17

from Sungai Rayu, Matang, Kuching, Sarawak

3.3 Type ofbacteria and their morphologies on P ALCAM selective agar 18

3.4 Sequences of primers used for specific Polymerase Chain Reaction 21

reaction

3.5 List ofantimicrobial agents for antimicrobial susceptibility test 22

4.1 Number of wildlife hosts tested for the occurrence of Listeria spp. 26

in Matang Wildlife Center, Kuching, Sarawak

4.2 List of samples and samplings method applied during the field trip 26

from Matang Wildlife Centre, Kuching, Sarawak

4.3 Gram staining on isolates from Matang Wildlife Centre, Kuching 29

and Sungai Rayu, Matang, Kuching, Sarawak

4.4 Percentage of resistant and susceptible strains towards the 33

antimicro bial agents

4.5 Antibiotic resistant patterns and MAR index of 10 confirmed 35

L. monocytogenes isolates

VI

LIST OF FIGURES

Figures Page

3.1 Location of the sampling district at Matang Wildlife Centre, 15

Kubah National Park and Sungai Rayu, Matang

4.1 Listeria spp. growth on P ALCAM agar 27

4.2 Colonies ofListeria spp. on Tryptose soy agar (TSA) 27

4.3 Listeria spp. appearances under light microscope 28

4.4 Amplicon obtained from Specific PCR for 10 L. monocytogenes isolates 31

from Kubah National Park, Kuching with virulence gene, hlyA

4.5 Percentage of resistant and susceptible strains towards the 34

antimicrobial agents

Vll

, .

LIST OF ABBREVIATIONS

J'L

bp

C

DNA

dNTP

G

L ;nnocua

L ;vanovii

L monocytogenes

L seeliger;

LLO

MgCh

mL

mm

mM

Mpl

NA

N.CI

PBS

PC-PLC

PCR

PI-PLC

RNA

Rpm

Spp.

TBE

TSA

TSB

UV

V

Microlitre

Base pair(s)

Cytosine

Deoxyribonucleic acid

Deoxyribonucleotide triphosphate

Guanine

Listeria innocua

Listeria ivanovii

Listeria monocytogenes

Listeria seeligeri

Listeriolysin 0

Magnesium chloride

Millilitre

Millimetre

Millimolar

Metalloprotease

Nutrient agar

Sodium chloride

Phosphate Buffer Saline

Phosphatidycholine-specific phospholipase C

Polymerase chain reaction

Phosphatidylinositol-specific phospholipase C

Ribonucleic acid

Revolutions per minute

Species

TrislBorate/EDT A

Tryptic soy agar

Tryptic soy broth

Ultra violet

Voltage

VIll

· .

Detection ofL. monocytogenes in animal and water samples in Kubah National Park, Kuching, Sarawak

AiOO Syazana Anuar

Resource Biotechnology Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Listeria monocytogenes (L. monocytogenes) is facultative anaerobic pathogens that can asymptomatically infect various animals. Normally, it also can be found in environment such as vegetation, water, soil, dust, mud and sewage. This study was conducted to detect the presence of L. monocytogenes and antimicrobial susceptibility characteristic from wildlife and water samples at Kubah National Park, Kuching. Samples collected were incubated and streaked on selective medium P ALCAM agar to confirm the presence of Listeria spp. before they were further tested using biochemical and molecular analysis. Specific Polymerase Chain Reaction (PCR) assay were performed to target specific virulence gene, such as haemolysin gene, h/yA to further distinguish the presence of this pathogenic bacteria in the sample. The h/yA gene plays major roles in haemolytic properties in L. monocytogenes as it produces Listeriolysin 0 (LLO) which involve actively in lysis process of phagocytic membrane by releasing pore-forming toxin. Overall, out of the 30 samples tested, 10 samples were confirmed as to contain L. monocytogenes isolates and selected to subsequent antimicrobial susceptibility test. The test revealed that 90010 of the isolates were resistant to Tetracycline (30 ~g) and Erythromycin (30 Jlg) while Gentamycin (10 Jlg) and Tobramycin (10 Jlg) has the lowest level of resistance (0%). The mUltiple antibiotic resistances shown by L. monocytogenes isolates in this study indicate the potential health hazard associated with the possible transmission between wildlife and water to its surrounding environment especially visitors of Kubah National Park, Kuching

Keywords: L. monocytogenes, Kubah National Park, Polymerase Chain Reaction (PCR), h/yA gene, Antimicrobial susceptibility

ABSTRAK

Listeria monocytogenes (L. monocytogenes) adalah bakteria fakultatif anarobik yang boleh menjadikan haiwan sebagai pembawa asimptomatik. Ia juga dijumpai di persekitaran seperti tumbuhan, air, tanah, debu, lumpur dan kumbahan. Kajian telah dijalankan untuk mengesan kehadiran L. monocytogenes dan ciri ketahanan antibiotik dari sampel hidupan liar dan air di Taman Negara Kubah, Kuching. Sampel yang diperolehi telah diinkubasi dan digores di atas medium selektif, agar P ALCAM untuk mengesahkan kehadiran Listeria spp. Tindak balas rantai polimerase (PCR) spesifik telah dilakukan untuk mengesan gen virulens spesifik, seperti hemolysin gen, h/yA untuk mengesabkan lagi kehadiran bakteria patogenik ini. Gen h/yA memainkan peranan penting dalam sifat haemolitik L. monocytogenes kerana ia menghasilkan listeriolysin 0 (LLO) yang terlibat secara aktif di dalam proses lisis di membran berfagosit dengan membebaskan toxin penghasilliang. Keseluruhannya, dari 30 sampel yang telah diuji, hanya 10 sampel telah disahkan sebagai L. monocytogenes dan dipilih untuk ujian ketahanan antimikrobial berikutnya. Ujian mendedahkan bahawa 90% daripada isolat mempunyai ketahanan terhadap Tetracycline (30 Jlg) dan Erythromycin (30 Jlg) manakala Gentamycin (10 ~g) dan Tobramycin (10 Jlg) mempunyai tahap rintangan terendah (0%). Beberapa ketahanan yang ditunjukkan oleh L. monocytogenes dalam kajian in.i menunjukkan bahawa terdapat potensi bahaya kepada kesihatan dengan kebarangkalian jangkitan di antara bidupan liar dan air kepada persekitaran terutama pengunjung Taman Negara Kubah, Kuching.

Kata kund: L. monocytogenes, Taman Negara Kubah, Tindak balas rantai polimerase (PCR), Gen h/yA . Ketahanan antimikrobial

IX

· ., ,.

CHAPTER I

INTRODUCTION

Listeria spp. are gram positive, non-sporing, non-encapsulated and motile bacteria. They

were classified under aerobic and facultative anaerobic. Microscopically, Listeria spp.

usually appears as short rods with rounded ends singly or short chain. The genus Listeria is

characterized into six species; with Listeria monocytogenes and Listeria ivanovii are

classified as pathogenic species while other type of Listeria spp. are considered as avirulent

and seldom to cause any disease (Mainou-Fowler, 1988). According to Center for Disease

Control (CDC), the latest incidence reported was on 1st November 2011 involving 139

victims infected at United States. L. innocua is considered as an indicator bacterium for the

presence of L. monocytogenes as it constantly appears in various environmental samples

together, while L. ivanovii rarely involved in human pathology, and L. seeligeri has been

reported only once to be the cause of meningitis in a nonimmunocompromised adult (Lovett

and Twedt, 1988).

According to Morse (1994), cited by Daszak (2000), translocation and introduction of

animals to new geographic regions correspond to increased human global travel and

commerce as underlying factors for infectious disease emergence. Nowadays, there are trends

of emergence of pathogenic bacteria from wildlife (Dazdak, 2000). Animals in wildlife hold

possibility of acting as reservoir and posse threat to global biodiversity and human

population. This is due to population expansion which will lead to human invasion and

risking themselves towards pathogenic bacteria. Intestinal tract of animal such as wild and

feral mammals, crustaceans, birds and fish act as reservoir of infection. Besides humans, at

1

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· .

least 42 species of wild and domestic mammals, rodents and 17 avian species, including

domestic and game fowl, can act as a vector host for L. monocytogenes (Todar, 2003; Lloyd,

2008).

Other environmental samples such as water and sediments from Sungai Rayu were

also been included in the study to measure the rate of occurrence in the surrounding area

around Kubah National Park, Kuching. Molecular technique such as Polymerase Chain

Reaction (PCR) used for virulence gene detection after the preliminary and identification

method. One of the advantages of this technique are they offer accurate, reliable and time

saving procedure to obtain result. Specific PCR is used in this experiment where single

primer set allowed detection and amplification of gene of interest in single run test. This

molecular technique give ability to target specific virulence gene, such as hemolysin gene,

hlyA in L. monocytogenes to further acknowledge the presence of this pathogenic bacteria in

the sample (Coco lin & Rantsiou, 2009). Multiple keys virulence factors are important in L.

monocytogenes pathogenesis such as hemolysin (hlyA), phosphatidylinositol phospholipase C

(PIcA), actin polymerization protein (actA) and invasive associated protein (iap) (Furrer et al.

1991, Portnoy et al. 1992 as cited in Kuhn and Goebel (2007». However, the hlyA gene is

used as a PCR target as it is species-specific to identify L. monocytogenes (Blais et al., 1994).

The objective of this study is to investigate the occurrence of L. monocytogenes at

Kubah National Park by analyzing wildlife faeces waste and swab samples from their anal,

cloacal, faecal wastes. Environmental samples such as water and sediments were also been

analyzed. This area is chosen as it is one of the popular areas and received many visitors each

year for recreational purpose such as camping and hiking. At this level, there is possibility of

direct transmission ofListeria spp. especially L. monocytogenes from wildlife to human. The

2

t • , ;

research is done by detecting the presence ofvirulence genes hlyA from the samples collected

using Polymerase Chain Reaction (PCR) assay. Meanwhile the phenotypic characterization is

based on biochemical test and antibiotic resistance pattern. Antibiotic sensitivity test is one of

the common methods being used to differentiate L. monocytogenes according to its serovars

(Yeh, 2004).

3

, .

CHAPTER II

LITERATURE REVIEW

1.1 Selection of species studied

1.1.1 Family Listeriaceae

The first published article describing about L. monocytogenes was done by Murray et al.

(1926) after observing six cases of sudden death of young rabbit in 1924 at animal breeding

establishment of the Department of Pathology, Cambridge. Previously genus Listeria was

included in the Corynebacteriaceae family in 1930 after not listed in three early edition of

Bergey's Manual of Determinative Bacteriology. Nevertheless, in 1974 edition, Listeria is

considered to have uncertain similarity with Corynebacteriaceae, thus it was listed with

Erysipelothrix and Caryophanon in Lactobacillaceae family (Bergey' s Manual of

Determinative Bacteriology, 8th Edition, 1974 as cited by Rocourt and Buchrieser, 2007).

In 1975, Wilkinson and Jones proposed that Listeria, Gamella, Bronchotrix,

Lactobacillus and Streptococcus to be classified in Lactobacillaceae family. This was based

on the numerical taxonomy analysis describing phenotypical features as the characterization

precursor in classification of genus Listeria. The position of Listeria was further studied in

1977 by Wilkinson and Jones and Feresu and Jonese in 1988. 16S rRNA cataloging studies of

Stackebrandt et al. (1983) confirmed that L. monocytogenes was a dissimilar taxon within the

Lactobacillus-Bacillus branch of the bacterial phylogeny. Recently, the distance relationship

between Lactobacillus and Listeria is further confmned when 23S rRNA is sequenced. The

4

Pusat Khidmat Maldumat Abdemik I •

UNIVERSm MALAVSIA SARAWAK

result shows that Listeria exhibit greater similarity to Bacillus and Staphylococcus (Sallen et

al., 1996).

Presently genus Listeria is classified in Clostridium sub branch between Lactobacillus

and Bacillus due to its low proportion of G+C in its genome. They are proved to be distantly

related to Streptococcus, Lactococcus, Enterococcus, Staphylococcus, Kurthia, Gemella, and

ErySipelothrix. This also applies to genus Brochothrix as it is closely related to Listeria

(Wilkinson and Jones, 1977; Feresu and Jones, 1988 as cited by Ryser and Marth, 2007).

2.1.2 Genus Listeria

Genus Listeria contains only L. monocytogenes during its first discovery. In 1948, L.

denitrificans was added because of its ability to reduce nitrate. However, it was transferred to

new genus, Jonesia. Rocourt et al. (1987) prove that this species is not part of Listeria family

as l6S rRNA result shows that it belongs to corneform group ofbacteria rather than Listeria.

L. grayi was added to the genus in i 966 followed by L. murrayi. However, due to similarities

in numerical taxonomy analysis, multilocus enzyme analysis and DNAIDNA hybridization

analysis result, former L. murrayi is considered as part ofL. grayi too (Stuart and WeIshimer,

1974). Evidence proved that they share same number of chemotaxonomic properties, base

composition values, pattern in cellular fatty acid, fatty aldehyde and composition of

lipoteichoic acids (Roucourt and Buchrieser, 2007). L. innocua was added to the family in

1981 followed by L. wishmeri and L. seeligeri in 1983 and last species to be added to the

genus was L. ivanovii in 1985. Each species in genus Listeria contain gene conservation

among of them and each combination of genes (either some of the gene present or absent)

will further define the species itself (Rocourt and Buchrieser, 2007).

5

I •

Genomic group I consist ofL. monocytogenes belong to serovars 1I2a, 1I2b, 1I2c,3a,

3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7 while genomic group 2 contain L. ivanovii fit into

serovars 5 and considered to be strongly haemolytic strains. L. ivanovii is further divided into

two, L. ivanovii subspecies ivanovii which is ribose positive while L. ivanovii subspecies

londoniensis consist ofribose negative. L. innocua corresponded into genomic group 3 which

contain nonhaemolytic and non-pathogenic strains of 4ab, 6a, 6b. L. welshmeri is classified

into genomic group 4 with nonhaemolytic strains for mice of serovars 6a and 6b. Genomic

group 5 consist of haemolytic and non-pathogenic strains Il2b, 4c, 4d, 6b and several

undesignated serovars. This group is called L. seeligeri in 1983 (Roccourt et aI., 1982).

2.2 L monocytogenes

2.2.1 Morphology and Culture

L. monocytogenes is gram positive bacteria appear with rod and rounded end microscopically.

It is the main agent in Listeriosis disease. Usually the cell can be found in either in single

units or V, Y or palisade cluster arrangement form. Genus Listeria appears to be small with

0.5 Ilm in diameter and I to 2 Ilm in length. It is motile from its few peritrichous flagella

when cultured in room temperature; 20 to 25°C (Parihar, 2004). According to Robbins and

Theriot (2003), Listeria usually rotates around its long axis with help from actin based

motility, with average time per rotation is 507±I06 micrometer per second and average

distance per rotation is 29.4± 11.8 micrometer. Listeria colonies appear to be able to survive

in low temperature condition (1 to 2 °C to 45°C), various pH condition; minimum pH 4.3,

optimum pH 6.8, maximum pH 9.6 and can tolerate high salt concentration condition

(Roucourt and Buchrieser, 2007).

6

· . L. monocytogenes is non-sporing and non-encapsulated bacteria. Normally it will

grow well in bacteriological media, especially in presence of fermentable sugar such as

glucose. It colonies usually appear in blue green when cultured in laboratory, with smooth

colonies appearance. The size of colony usually begins between 0.2 to 0.8 mm in diameter

and will expand exponentially up to 5 mm or more in well separated plate. Occasionally

rough colonies will be formed and this process is not reversible between each other (Parihar,

2004).

In culture, L. monocytogenes exhibits tumbling motility properties when it has been

stabbed in semi-solid motility agar incubated at 20 to 25°C. The cells show random twisting

movement with unusual rotation before moving into various directions. Furthermore, the

colony will show haemolytic properties which will differentiate L. monocytogenes colony

from some other type of Listeria species. "Umbrella" or inverted "pine tree" like growth is

observed to appear below 3 to 5 mm the surface due to cells' microaerophilic properties in

stabbed cultures. Listeria colony usually will produce carboxylic acids, hydroxyl acids and

alcohols that will cause pungent acid odour in culture (Roucourt and Buchrieser, 2007).

1.3 Factors affecting the growth of L. monocytogenes

1.3.1 pH

The growth of L. monocytogenes was only observed in the culture at pH 5.0 and pH 6.0 in

respective temperature of 10 °C and 5 °C (McClure et at., 1991). According to a study

conducted by Conner et at. (1986) range of pH that initiate growth was 5 to 5.7 at 4.0 °C

while in 30°C, the pH varies 4.3 to 5.2. Several studies also recorded that L. monocytogenes - ~

can growth at pH 4.4 in 30°C within 7 days (George et at., 1988); pH 4.5 in 19°C using

7

trypic soy broth (Buchanan and Klawitter, 1991) and pH 4.66 in 30°C for 66 days by

Colburn et al. (1990). A recent study by Roucourt and Buchrieser (2007) stated that L.

monocytogenes culture can grow in optimum pH 6.8 and able to survive in smallest pH 4.3

and pH 9.6 as the limit.

2.3.2 Temperature

According to Juntilla et al. (1988), the mean mmlmum temperature suitable for L.

monocytogenes growth was between 1.1 °C ± 0.3 °C with range from 0.5 °C to 3.0 °C.

Among other species ofListeria spp., L. monocytogenes is seen to have the ability to survive

in temperature in 0.6 °C lower minimum temperature than the other species. It is suggested

that haemolysin actually enhance the chance of survival properties of culture in cold

environment (Jay, 1992). L. innocua, L. grayi, L. murrayi and L. welshimeri has minimum

temperature range 1.7 °C to 3.0 °C with mean around 1.7 °C ± 0.5 °C (Juntilla et al., 1988).

Listeria culture can survive until temperature around 45°C before the amount of growth start

to decrease constantly as the temperature increase.

2.4 Virulence factors

AImng six species, only two types of Listeria are further recognized to be pathogenic; L.

monocytogenes, for causing listeriosis for human and animal while L. ivanovii is virulence

towards mice. The pathogenic factors depend on the expression of virulence gene and

immune level of individual. According to Gouin et al. (1994), L. monocytogenes causes

severe infections that primarily affect immunocompromised people, pregnant women, and

neonates but occasionally also healthy people. Its ability to infect internal organ; ability of

8

membrane

"

crossing intestinal was recorded by Macro et al. (1997) followed by blood brain barrier

(Uldry et aI., 1993; Berche, 1995) and transplacental transmission (Gray and Killinger, 1993;

Lecuit et a/., 2004, as cited by Parihar, 2004).

Several genes have been identified to be involved in L. monocytogenes invasion.

Almost all the virulence gene actually situated in so-called prfA-dependent virulence gene

cluster, however there are several genes which are not included in the cluster also connected

to the pathogenic factor in L. monocytogenes. In prfA cluster, there are six well characterized

gene, prfA, pleA, hly, mpl, actA and picE. These genes encode several products that are

actively involve such as listeriolysin 0 (LLO) which is encoded by hly gene,

phosphatidylinositol-specific phospholipase C (PI-PLC) produced by pleA, a

phosphatidycholine-specific phospholipase C (PC-PLC) encoded by picE, metalloprotease

(Mpl) by mp/ protein involved in actin polymerization named actA, encoded by actA gene

and positive regulatory factor prfA encoded by prfA gene (Kuhn and Goebel, 2007).

L. monocytogenes, L. seeligeri and L. ivanovii are species in genus Listeria that shows

haemolytic characteristic; although it is proven that only L. monocytogenes and L. ivanovii

are pathogenic. The hly gene plays major roles in haemolytic properties in L. monocytogenes.

It produces listeriolysin 0 (LLO), which actively involved in lysis process of phagocytic

by releasing pore-forming toxin. This 60-kDa protein actually helps L.

monocytogenes to escape from host cell's vacuole and infected other cell nearby. Two

phospholipases C are also involved during invasion and spread stages. PI-PLC involve in

primary phaglysosome while PC-PLC assist in formation of secondary phagolysosome

(parihar, 2004).

9

According to Domann et al. (1992) and Kocks et al. (1992), actA act as listeria

surface protein that allows actin-bases intracellular motility. actA helps cell to propel forward

by assembling and activate host actin cytoskeletal molecules. This movement occur without

leaving the intracellular environment, thus host immune response will not be triggered. In

addition, another important element involve in L. monocytogenes virulence is bile salt

hydrolase (BSL) enzyme. According to Parihar (2004), BSL is encoded by bsh gene will

protect L .monocytogenes colony from bile salt toxicity and assist in hepatic and intestinal

listeriosis phase.

1.S Epidemiology

Based on World Health Organization (2012) definition, epidemiology is the study of the

distribution of occurrence and pattern of outbreak and their causes within well defmed

population. Overall study of interaction between organism and their host in the same or

different population give advantage in better understanding of the resistance variation and

control the spreading of disease. Zoonotic infections can be defmed as type of infection that

originate from animals and can be transmissible to human. L. monocytogenes, which will

cause Listeriosis is listed among zoonotic transmitted diseases worldwide either among

animal and human. They were capable to survive in many environment conditions according

to soil types, especially damn surface soil and presence of decaying vegetation source

(Sauders and Wiedmann, 2007).

Transmission of Listeria spp. to animals, especially wildlife is still considered not

atcIDsively being studied compared to domestic livestock an~ food source due to !he fact that

.LI8terla is strongly considered as food borne pathogen and predominantly transferred through

10

population

, .

non-zoonotic infection (Seeliger, 1961). However, animal has potential to act as reservoir

vector host in manifesting the pathogenic bacteria before transmits it through food, water

source or any direct contact to human. This kind of transmission is not only applicable to

domestic animals but applies to wild animals as well (Goldsmith, 2005). There is probability

that infection occur from animal feeding habits, followed by harbouring the pathogen inside

its own body and shedding it in their faecal and nasal waste. According to Gray and Killinger

(1966), the mode of transmission of Listeria spp. among avian is poorly studied. However

evidence showed that bacteria were transmitted through feeding behaviour such as pecking

on contaminated soil, fecal material or dead mammals. Fenlon (1985) stated that there was

significant increase of ovine listeriosis in nesting season which likely associate with

consumption of silage contaminated with Listeria spp.

2.6 Listeriosis

L. monocytogenes which often regarded as food borne pathogen causing Listeriosis hold

possibility of being transmitted not only limited to food source but through environmental

too. Listeriosis incident has been reported worldwide since 1924. Although Listeriosis cases

occur rarely, the rate of severe illness and fatalities reported should be taken into

consideration. Normally among human, L. monocytogenes will infect immunocompromised

and transmitted either through direct contact or from consumption of

contaminated raw foods such as cheese and milk (Painter and Slutsker, 2007).

However in animal, there are difference in mode of transmission for the infection to

occur such as by feeding on contaminated source of food or water source, sea~on, immune

status; pregnancy and transmission between mother and foetus and breed of livestock in farm

11

parasites that able

peater risk ofdeath.

animals. Nonnally Listeriosis will cause encephalitis that win lead to several stages of

meningoencephalitis in animal. Encephalitis is the infection 0 f central nervous systems and

can be indicated by the condition where body temperature increase in and animal refuses to

eat or drink followed closely by neurological disturbance. This disturbance will further

damage animals own coordination and sometime will cause tremor (Wesley, 2007). In fowl

and wild birds, most common manifestation of Listeriosis is septicaemia which is the

formation of necrosis at liver and spleen (Gray et ai., 1966). Diarrhoea and emaciation are

also common in infected animals. As a result, they will shed L. monocytogenes in their faecal

and liquid waste, thus further facilitating the transmission of L. monocytogenes in

environment nearby (Wesley, 2007).

2.7 Antibiotic resistance

Antibiotic resistance is defined by World Health Organization (WHO, 2012) as the

characteristic of ability of microorganism that previously susceptible to become resistance to

the particular antimicrobial medicine. This microorganism includes bacteria, virus and some

to withstand inhibition attack from antimicrobial medicines such as

antibiotic, antiviral and antimalarial. As a result, resistant strains often failed to give success

result to therapeutic treatment resulting prolonged time of infection and exposed the host to

According to Raff et ai. (1984), resistance to Ampicilin, which is the first

recommended drug against Listeria was fIrst observed in 1984. It was suggested that one of

main reasons of this resistance to develop are due to the constant exposure of antibiotic

fi>od routes and over prescription over one antimicrobial agent. L. monocytogenes is also

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reportedly to have the most frequent resistance towards Tetracycline. It was identified that the

three self-transferable multiresistant plasmids from L. monocytogenes iso lated from humans

carried the tet(S) gene that caused resistance towards Tetracycline (Radom et al., 1993). At

present, resistance ofListeria spp. is still seen slow as most of them are still quite susceptible

to most of the recommended treatments. Based on Okada et al. (2011), this trend however

needs to be observed carefully and continuous surveillance towards resistance in Listeria spp.

need to be done since the antimicrobial chemotherapy is the only current treatment of

Listeriosis in human and animals.

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CHAPTERUI

MATERIALS AND METHOD

3.1 Sample collections

Samples were collected by a team of researchers from Molecular Microbiology Lab from

Universiti Malaysia Sarawak (UNIMAS) during a field trip to Matang, Kuching. Two

sampling sites had been chosen which was Matang Wildlife Center for animal samples

collection and Sungai Rayu for water sampling site. A total of 30 samples, where 22

animals samples were sampled from wildlife (bats, tree shrew, squirrels, mongoose and

birds) while 8 environmental samples (water and sediments) were collected and shown in

Table 3.1. Matang Wildlife Centre situated in Kubah National Park consists of large

ecosystem area for endangered wildlife located 35 km from Kuching while Sungai Rayu is

a river situated along the human settlement area, Kampung Rayu. The map showing the

location ofsampling are shown in Figure 3.1

Bats and birds samples were captured using mist nets while rodents, mongoose and

tree shrew were captured using cage traps. Animal samples were acquired using swabbing

method where anal swabs and cloacal swabs were collected using sterile cotton swab. For

faeces and water samples, the specimens were collected directly from the respective

sources. All specimens were immediately placed into 1 mL of Phosphate Buffer Saline

(PBS) and stored at 4 °C inside the ice box throughout the field trip. Samples were

immediately processed at laboratory within the same day of sampling to avoid sample

defJradation and contamination.

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+-__ Sungai Rayu, Matang

I ,/

+--- Mat g Wildlife Ce

"

FIgure 3.1 Location of the sampling district at Matang Wildlife Centre, Kubah National Park and Sungai Rayu, Matang (Source: Google Map, 2012).

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